Conjugation process of bacterial polysaccharides to carrier proteins

ABSTRACT

Process for conjugation of bacterial saccharides including  Streptococcus pneumoniae  and  Haemophilus influenzae  saccharides by reductive amination are provided herein.

The present invention relates to a process for conjugation. Inparticular, it relates to the conjugation of saccharides and proteinsusing reductive amination.

BACKGROUND

Bacterial capsular polysaccharides have been widely used in immunologyfor many years for the prevention of bacterial disease. A problem withsuch a use, however, is the T-independent nature of the immune response.These antigens are thus poorly immunogenic in young children. Thisproblem has been overcome through conjugating the polysaccharideantigens to a carrier protein (a source of T-helper epitopes) which maythen be used to elicit a T-dependent immune response, even in the firstyear of life.

Various conjugation techniques are known in the art. Conjugates can beprepared by direct reductive amination methods as described in,US200710184072 (Hausdorff) U.S. Pat. No. 4,365,170 (Jennings) and U.S.Pat. No. 4,673,574 (Anderson). Other methods are described inEP-0-161-188, EP-208375 and EP-0-477508. The conjugation method mayalternatively rely on activation of hydroxyl groups of the saccharidewith 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to forma cyanate ester. Such conjugates are described in PCT publishedapplication WO 93/15760 Uniformed Services University and WO 95/08348and WO 96/29094. See also Chu C. et al Infect. Immunity, 1983 245 256.

Reductive amination involves two steps, (1) oxidation of the antigen,(2) reduction of the antigen and a carrier protein to form a conjugate.The oxidation step may involve reaction with periodate, howeveroxidation by periodate may lead to size reduction (WO94/05325).

SUMMARY OF INVENTION

The inventors have surprisingly found that using lower concentrations ofperiodate in the presence of low phosphate may lead to retention of sizeand/or the retention of epitopes.

In a first aspect of the invention there is provided a process forconjugating a bacterial saccharide(s) comprising the steps of

-   -   a) reacting the bacterial saccharide with 0.001-0.7, 0.005-0.5,        0.01-0.5, 0.1-1.2, 0.1-0.5, 0.1-0.2, 0.5-0.8, 0.1-0.8, 0.3-1.0        or 0.4-0.9 molar equivalents of periodate to form an activated        bacterial saccharide;    -   b) mixing the activated bacterial saccharide with a carrier        protein;    -   c) reacting the activated bacterial saccharide and the carrier        protein with a reducing agent to form a conjugate;        -   or    -   a) reacting the bacterial saccharide with 0.001-0.7, 0.005-0.5,        0.01-0.5, 0.1-1.2, 0.1-0.5, 0.1-0.2, 0.5-0.8, 0.1-0.8, 0.3-1.0        or 0.4-0.9 molar equivalents of periodate to form an activated        bacterial saccharide;    -   b) mixing the activated bacterial saccharide with a linker;    -   c′) reacting the activated bacterial saccharide with the linker        using a reducing agent to form a bacterial saccharide-linker;    -   d) reacting the bacterial saccharide-linker with a carrier        protein to form a conjugate;        wherein step a) occurs in a buffer which does not contain an        amine group, and the buffer has a concentration between 1-100        mM.

In a second aspect of the invention there is provided a conjugateobtainable by the process of the invention.

In a third aspect of the invention there is provided a conjugateobtained by the process of the invention.

In a fourth aspect of the invention there is provided an immunogeniccomposition comprising the conjugate of the invention and apharmaceutically acceptable excipient.

In a fifth aspect of the invention there is provided a vaccinecomprising the immunogenic composition of the invention.

In a sixth aspect of the invention there is provided a use of theimmunogenic composition of the invention or the vaccine of the inventionin the prevention or treatment of bacterial disease

In a seventh aspect of the invention there is provided a use of theimmunogenic composition of the invention or the vaccine of the inventionin the preparation of a medicament for the prevention or treatment ofbacterial disease.

In a eighth aspect of the invention there is provided a method ofpreventing or treating bacterial infection comprising administration ofthe immunogenic composition of the invention or the vaccine of theinvention to a patient.

In an ninth aspect of the invention there is provided an activatedbacterial saccharide, wherein the activated bacterial saccharidecomprises a repeat unit of formula (I):

wherein the activated bacterial saccharide comprises n repeat units andn is between 2 and 2400, between 500 and 2000, between 750 and 1500,between 1000 and 2000 or between 1500 and 2300.wherein at least 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 5%, 10% or 30% butless than 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 30% or 50% of S1is

and the remainder is

wherein S2 is either

and wherein S3 is either

DESCRIPTION OF FIGURES

FIG. 1. Size of 23F and 6B polysaccharides following periodatetreatment. The line marked with triangles shows the size of 6B in 10 mMphosphate buffer, the line marked with diamonds shows the size of 23F in10 mM phosphate buffer and the line marked with squares shows the sizeof 23F in 100 mM phosphate buffer.

FIG. 2. Comparison of immunogenicity of 23F conjugates using either CDAPor reductive amination conjugation. Graph a) describes the results of anELISA assay. Graph b) describes the results of an opsonophagocytosisassay.

FIG. 3 Evaluation of the immunogenicity of PS06B-CRM conjugated usingthe conjugation methods described in example 4 in a Balb/c mouse model.

FIG. 4 Evaluation of the immunogenicity of PS06B-CRM conjugated usingthe conjugation methods described in example 4 in a guinea pig model.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an improved process for conjugating an antigento a carrier protein. In particular, the invention provides a processfor conjugating a bacterial saccharide(s) comprising the steps of

-   -   a) reacting the bacterial saccharide with 0.001-0.7, 0.005-0.5,        0.01-0.5, 0.1-1.2, 0.1-0.5, 0.1-0.2, 0.5-0.8, 0.1-0.8, 0.3-1.0        or 0.4-0.9 molar equivalents of periodate to form an activated        bacterial saccharide;    -   b) mixing the activated bacterial saccharide with a carrier        protein;    -   c) reacting the activated bacterial saccharide and the carrier        protein with a reducing agent to form a conjugate;        -   or    -   a) reacting the bacterial saccharide with 0.001-0.7, 0.005-0.5,        0.01-0.5, 0.1-1.2, 0.1-0.5, 0.1-0.2, 0.5-0.8, 0.1-0.8, 0.3-1.0        or 0.4-0.9 molar equivalents of periodate to form an activated        bacterial saccharide;    -   b) mixing the activated bacterial saccharide with a linker;    -   c′) reacting the activated bacterial saccharide with the linker        using a reducing agent to form a bacterial saccharide-linker;    -   d) reacting the bacterial saccharide-linker with a carrier        protein to form a conjugate;        wherein step a) occurs in a buffer which does not contain an        amine group, and the buffer has a concentration between 1-100        mM.

The term ‘periodate’ includes both periodate and periodic acid. Thisterm also includes both metaperiodate (IO₄ ⁻) and orthoperiodate (IO₆⁵⁻), however in one particular embodiment the periodate used in themethod of the invention is metaperiodate. The term ‘periodate’ alsoincludes the various salts of periodate including sodium periodate andpotassium periodate. In one embodiment the periodate used is sodiummetaperiodate. When an antigen reacts with periodate, periodate oxidisesvicinal hydroxyl groups to form carbonyl or aldehyde groups and causescleavage of a C—C bond. For this reason the term ‘reacting an antigenwith periodate’ includes oxidation of vicinal hydroxyl groups byperiodate.

For the purposes of the invention an ‘activated bacterial saccharide’ isa bacterial saccharide which has been activated by step a) of theprocess of the invention.

For the purposes of the invention the term ‘conjugate’ indicates abacterial saccharide linked covalently to a carrier protein. In oneembodiment a bacterial saccharide is linked directly to a carrierprotein. In a second embodiment a bacterial saccharide is linked to aprotein through a spacer/linker.

The buffer used in step a) is a buffer which does not contain an aminegroup. In one embodiment the buffer is selected from the list consistingof phosphate buffer, borate buffer, acetate buffer, carbonate buffer,maleate buffer and citrate buffer. In a second embodiment the buffer isan inorganic buffer. The term inorganic buffer includes any buffersolution wherein the buffering capacity is due to the presence of acompound which does not contain carbon. Inorganic buffers of theinvention include phosphate buffer and borate buffer. In one embodimentthe buffer is phosphate buffer.

In one embodiment the buffer has a concentration between 1-100 mM, 5-80mM, 1-50 mM, 1-25 mM, 10-40 mM, 1-10 mM, 5-15 mM, 8-12 mM, 10-20 mM,5-20 mM, 10-50 mM, around 10 mM or around 20 mM. In a further embodimentthe pH in step a) is pH 2.5-8.0, pH5.0-7.0, pH 5.5-6.5, pH 5.8-6.3, oraround pH 6.0.

The term “saccharide” throughout this specification may indicatepolysaccharide, techoic acid or oligosaccharide and includes all three.It may indicate lipopolysaccharide (LPS) or lipooliogosaccharide (LOS).Before use Polysaccharides may be isolated from a source strain orisolated from the source strain and sized to some degree by knownmethods (see for example EP497524 and EP497525; Shousun Chen Szu etal.—Carbohydrate Research Vol 152 p 7-20 (1986)) for instance bymicrofluidisation. Oligosaccharides have a low number of repeat units(typically 5-30 repeat units) and are typically hydrolysedpolysaccharides.

In one embodiment the bacterial saccharide is a bacterial capsularsaccharide. In one embodiment of the present invention the bacterialsaccharide originates from Group B Streptococcus, Vibrio cholera,Streptococus pneumoniae (S. pneumoniae), Haemophilus influenzae (H.influenzae), Neisseria meningitidis (N. meningitidis), Staphylococcusaureus (S. aureus), enterococci, Salmonella Vi, or Staphylococcusepidermidis (S. epidermidis). In a further embodiment the bacterialsaccharide originates from S. pneumoniae, H. influenzae, N.meningitidis, S. aureus, enterococci, Salmonella Vi, or S. epidermidis.In a yet further embodiment the bacterial saccharide is a bacterialcapsular saccharide selected from a list consisting of: N. meningitidisserogroup A (MenA), B (MenB), C (MenC), W135 (MenW) or Y (MenY), Group BStreptococcus group Ia, Ib, II, III, IV, V, VI, or VII, Staphylococcusaureus type 5, Staphylococcus aureus type 8, Salmonella typhi (Visaccharide), Vibrio cholerae, or H. influenzae type b. In one embodimentthe bacterial saccharide is a capsular saccharide from Streptococcuspneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F. In a furtherembodiment the bacterial saccharide is an S. pneumoniae capsularsaccharide selected from the group consisting of 5, 6B, 6A, 7F, 9V, 14,or 23F. Optionally the bacterial saccharide of the invention is an S.pneumoniae capsular saccharide 23F, 6B or 6A. In one embodiment thebacterial saccharide is an S. pneumoniae capsular saccharide 23F. In oneembodiment the bacterial saccharide is an S. pneumoniae capsularsaccharide 6B. In one embodiment the bacterial saccharide is an S.pneumoniae capsular saccharide 6A. In a yet further embodiment thebacterial saccharide is Haemophilus influenzae b (Hib) polysaccharide oroligosaccharide. In one embodiment the bacterial saccharide containsvicinal anti diols.

The bacterial saccharide may be either a native polysaccharide or mayhave been reduced in size by a factor of no more than x2, x4, x6, x8,x10 or x20 (for instance by microfluidization [e.g. by Emulsiflex C-50apparatus] or other known technique [for instance heat, chemical,oxidation, sonication methods]). In one embodiment the bacterialsaccharide is microfluidised before step a). Oligosaccharides may havebeen reduced in size substantially further [for instance by known heat,chemical, or oxidation methods].

For the purposes of the invention, “native polysaccharide” refers to abacterial saccharide that has not been subjected to a process, thepurpose of which is to reduce the size of the saccharide. Apolysaccharide can become slightly reduced in size during normalpurification procedures. Such a saccharide is still native. Only if thepolysaccharide has been subjected to techniques which reduce asaccharide in size would the polysaccharide not be considered native.

The weight-average molecular weight of a bacterial saccharide suitablefor conjugation by the process of the invention may be between 20 kDaand 2000 kDa, between 30 kDa and 1000 kDa, between 40 kDa and 500 kDa,between 50 kDa and 400 kDa, between 75 kDa and 300 kDa or between 1000kDa and 2000 kDa. In the case of the native 23F capsular saccharide fromS. pneumoniae, the average molecular weight of the native polysaccharideis between 750-1500 kDa or 1200-1300 kDa. In the case of the native Hibsaccharide, the average molecular weight of the native polysaccharide isbetween 100 and 250 kDa. The molecular weight or average molecularweight of a saccharide herein refers to the weight-average molecularweight (Mw) of the bacterial saccharide measured prior to conjugationand is measured by MALLS. The MALLS technique is well known in the art.For MALLS analysis of saccharides, two columns (TSKG6000 and 5000PWxI)may be used in combination and the saccharides are eluted in water.Saccharides are detected using a light scattering detector (for instanceWyatt Dawn DSP equipped with a 10 mW argon laser at 488 nm) and aninferometric refractometer (for instance Wyatt Otilab DSP equipped witha P100 cell and a red filter at 498 nm). MALLS analyses may be carriedout using a TSKGMPwxI and 50 mM Na/K PO4, 200 mM NaCl pH 7.0 as elutionbuffer with 0.75 ml/min using RI/DAWN-EOS detector. In an embodiment,the polydispersity of the saccharide is 1-1.5, 1-1.3, 1-1.2, 1-1.1 or1-1.05 and after conjugation to a carrier protein, the polydispersity ofthe conjugate is 1.0-2.5, 1.0-2.0. 1.0-1.5, 1.0-1.2, 1.5-2.5, 1.7-2.2 or1.5-2.0. All polydispersity measurements are generated by MALLS.

Treatment with periodate may lead to a reduction in the size of thebacterial saccharide (sizing effect). In one embodiment the process ofthe invention reduces this sizing effect. This is seen for the 23Fbacterial saccharide from Streptococcus pneumoniae (as in example 1).For this reason, in one embodiment the average molecular weight of abacterial saccharide of the invention is between 1-1100 kDa, 100-470kDa, 200-300 kDa, 600-1100 kDa or 800-1000 kDa after step a) (measuredby MALLS as described above). In one embodiment the average molecularweight of the 23F saccharide is between 100-470 kDa or 200-300 kDa afterstep a). In one embodiment the average molecular weight of the Hibbacterial saccharide is between 1 and 50 kDa or between 5 and 10 kDaafter step a).

The term “carrier protein” is intended to cover both small peptides andlarge polypeptides (>10 kDa). The carrier protein may be any peptide orprotein. It may comprise one or more T-helper epitopes. The carrierprotein may be tetanus toxoid (TT), tetanus toxoid fragment C, non-toxicmutants of tetanus toxin [note all such variants of TT are considered tobe the same type of carrier protein for the purposes of this invention],polypeptides comprising tetanus toxin T-cell epitopes such as N19(WO2006/067632), diphtheria toxoid (DT), CRM197, other non-toxic mutantsof diphtheria toxin [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida etal J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103and CRM107 and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.No. 4,950,740; mutation of at least one or more residues Lys 516, Lys526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat.No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed in U.S.Pat. No. 5,843,711] (note all such variants of DT are considered to bethe same type of carrier protein for the purposes of this invention),pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13),OMPC (meningococcal outer membrane protein—usually extracted from N.meningitidis serogroup B—EP0372501), synthetic peptides (EP0378881,EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussisproteins (WO 98/58668, EP0471177), cytokines, lymphokines, growthfactors or hormones (WO 91/01146), artificial proteins comprisingmultiple human CD4+ T cell epitopes from various pathogen derivedantigens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as N19protein (Baraldoi et al (2004) Infect Immun 72; 4884-7) pneumococcalsurface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337),toxin A or B of C. difficile (WO 00/61761), H. influenzae Protein D(EP594610 and WO 00/56360), pneumococcal PhtA (WO 98/18930, alsoreferred to Sp36), pneumococcal PhtD (disclosed in WO 00/37105, and isalso referred to Sp036D), pneumococcal PhtB (disclosed in WO 00/37105,and is also referred to Sp036B), or PhtE (disclosed in WO00/30299 and isreferred to as BVH-3).

In one embodiment of the invention the carrier protein is selected fromthe group consisting of: tetanus toxoid (TT), fragment C of tetanustoxoid, diphtheria toxoid (DT), CRM197, Pneumolysin (Ply), protein D,PhtD, PhtDE and N19. In a further embodiment the carrier protein isCRM197. In a still further embodiment the carrier protein is tetanustoxoid (TT).

In one embodiment step a) is carried out in the dark.

When an antigen reacts with periodate, periodate oxidises vicinalhydroxyl groups to form carbonyl or aldehyde groups and causes cleavageof a C—C bond. The oxidation step (step a)) may occur as describedbelow:

When low concentrations of buffer, in particular phosphate buffer andlow amounts of periodate are used, this may reduce the sizing effectdescribed above.

Streptococcus pneumoniae capsular saccharides contain vicinal hydroxylgroups which are capable of being oxidised by periodate as can be seenfrom the structures of the repeated regions shown below:

In one embodiment less than 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 5%, 10%,30% or 50% of the vicinal diols of the bacterial saccharide becomeoxidised during step a).

In one embodiment the carbonyl group produced in step a) reacts with anamine group on the carrier protein in step c). This may occur accordingto the following reaction scheme:

In one embodiment the bacterial saccharide is present at a concentrationof between 0.2 g/l and 14 g/l 8 g/l and 12 g/l, 10 g/l and 12 g/l, 1 g/land 4 g/l, 0.2 g/l and 1 g/l or between 0.4 g/l and 0.6 g/l or around 11g/l or around 0.5 g/l in step a). In one embodiment the initialconcentration of carrier protein in step b) is between 0.5 g/1 and 35g/l, 25 g/l and 35 g/l, 0.5 g/l and 5 g/l or between 0.8 g/l and 2 g/1or around 32 g/l or 1 g/l. In a further embodiment the initialconcentration of activated bacterial saccharide in step b) is between0.2 g/l and 20 g/l, 10 g/l and 28 g/l, or 0.2 g/l and 4 g/l or between 1g/l and 2 g/l or around 15 g/l or 1.6 g/l. In a further embodiment theinitial ratio of activated bacterial saccharide to carrier protein instep b) is 2.0:1 to 0.1:1, 1.8:1 to 0.4:1, 1.4:1 to 1.6:1, 1:1 to 1.4:1,1.8:1 to 1.6:1, 0.8:1 to 0.4:1, 0.7:1 to 0.5:1, or 0.7:1 to 0.6:1(w/w).In a further embodiment the final ratio of carrier protein to bacterialsaccharide after step c) or c′) is 0.5:1 to 4:1, 0.8:1 to 3.2:1, 0.5:1to 1.8:1, 1.4:1 to 1.8:1, 1:1 to 1.2:1 or 2.5:1 to 3.5:1.

In one embodiment the temperature of the reaction in step a) is 4-40°C., 10-32° C., 17-30° C. or 22-27° C. Typically this temperature ismaintained through step a). The reaction temperature during step c) is4-40° C., 10-32° C., 17-30° C. or 22-27° C. Typically this temperatureis maintained through step c).

In one embodiment step a) of the process of the invention takes place inless than 30 hours, between 5 and 25 hours, between 15 and 25 hours,between 30 minutes and 25 hours, between 1 hour and 35 hours, between 10and 20 hours, or between 15 and 20 hours around 18 hours or around 1hour. In one embodiment step c) of the process of the invention takesplace in between 10-60 hours, 10-20 hours, 20-60 hours, between 30-50hours, or between 35-45 hours.

Conjugation may also occur through the addition of a hetero- orhomo-bifunctional linker using the chemistry of the invention. One endof the linker will react with the activated antigen by reductiveamination, however the other end of the linker may react with thecarrier protein using any type of chemistry. For this reason the linkerwill contain at least one reactive amino group, if the linker ishomo-bifunctional it will contain two reactive amino groups, if thelinker is hetero-bifunctional it will contain one reactive amino groupand a different reactive group, in one embodiment this second reactivegroup is a reactive carbonyl group. In one embodiment the linker isbetween 1 and 20 Angstroms in length. In a further embodiment the linkerhas between 4 and 20, 4 and 12, or 5 and 10 carbon atoms. A possiblelinker is adipic acid dihydrazide (ADH). Other linkers includeB-propionamido (WO 00/10599), nitrophenyl-ethylamine (Geyer et al (1979)Med. Microbiol. Immunol. 165; 171-288), haloalkyl halides (U.S. Pat. No.4,057,685), glycosidic linkages (U.S. Pat. No. 4,673,574, U.S. Pat. No.4,808,700), hexane diamine and 6-aminocaproic acid (U.S. Pat. No.4,459,286).

In general the following types of chemical groups on the carrier proteincan be used for coupling/conjugation as the second reactive group:

A) Carboxyl (for instance via aspartic acid or glutamic acid). In oneembodiment this group is linked to an amino group on a linker withcarbodiimide chemistry e.g. with EDAC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)).

Note: instead of EDAC above, any suitable carbodiimide may be used.

B) Amino group (for instance via lysine). In one embodiment this groupis linked to a carboxyl group on a linker with carbodiimide chemistrye.g. with EDAC. In another embodiment this group is linked to hydroxylgroups activated with CDAP or CNBr on a linker; to linkers having analdehyde group; to linkers having a succinimide ester group.

C) Sulphydryl (for instance via cysteine). In one embodiment this groupis linked to a bromo or chloro acetylated linker with maleimidechemistry. In one embodiment this group is activated/modified with bisdiazobenzidine.

D) The protein could be modified to contain an alkynyl or azide group,this could be conjugated to the linker using the ‘click’ chemistry(described in Tetrahedron letters (June 2005) 46:4479-4482).

Note: instead of EDAC above, any suitable carbodiimide may be used.

Reducing agents which are suitable for use in the process of theinvention include the cyanoborohydrides, such as sodiumcyanoborohydride, borane-pyridine, or borohydride exchange resin. In oneembodiment the reducing agent is sodium cyanoborohydride. In oneembodiment between 0.5 and 2, 0.6 and 1.5 or 0.8 and 1.2 or around 1.0molar equivalent of sodium cyanoborohydride is used in step c). In afurther embodiment the reducing agent comprises sodiumtriacetoxyborohydride, in a further embodiment between 2 and 10 orbetween 3 and 9 molar equivalent or around 2.5 molar equivalent ofsodium triacetoxyborohydride is used in step c).

Before step c) the activated bacterial saccharide and the carrierprotein may be lyophilised. In one embodiment the activated bacterialsaccharide and the carrier protein are lyophilised together. This canoccur before step b), or after step b). In one embodiment thelyophilisation takes place in the presence of a non-reducing sugar,possible non-reducing sugars include sucrose, trehalose, raffinose,stachyose, melezitose, dextran, mannitol, lactitol and palatinit.

In a further embodiment the non-reducing sugar is selected from thegroup consisting of sucrose, trehalose or mannitol.

In one embodiment steps b) and/or c) are carried out in DMSO(dimethylsulfoxide) solvent. In a further embodiment steps b) and/or c)are carried out in DMF (dimethylformamide) solvent. The DMSO or DMFsolvent may be used to reconstitute the activated bacterial saccharideand carrier protein which has been lyophilised.

At the end of step c) there may be unreacted carbonyl groups remainingin the conjugates, these may be capped using a suitable capping agent.In one embodiment this capping agent is sodium borohydride (NaBH₄), forexample the product of step c) may be reacted with sodium borohydridefor 15 mins-15 hrs, 15 mins-45 mins, 2-10 hrs or 3-5 hrs, around 30 minsor around 4 hrs. In a further embodiment capping is achieved by mixingthe product of step c) with around 2 molar equivalents or between 1.5and 10 molar equivalents of NaBH₄.

The invention also provides a further step e) of purifying theconjugate, step e) may comprise diafiltration, for example diafiltrationwith a cut-off of 100 kDa. In addition or alternatively step e) maycomprise ion exchange chromatography. In a further embodiment step e)may comprise size exclusion chromatography. In one embodiment theprocess of claims 1-51 comprises a further step f), wherein theconjugate is sterile filtered.

The conjugate may also be mixed with further antigens. In one embodimentthe further antigens comprise at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 S. pneumoniae saccharides selected from the groupconsisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In one embodiment thefurther antigens comprise S. pneumoniae saccharides 4, 6B, 9V, 14, 18C,19F and 23F. In one embodiment the further antigens comprise S.pneumoniae saccharides 4, 6B, 9V, 14, 18C and 19F. In one embodiment thefurther antigens comprise S. pneumoniae saccharides 4, 9V, 14, 18C, 19Fand 23F. In one embodiment the further antigens comprise S. pneumoniaesaccharides 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. In one embodimentthe further antigens comprise S. pneumoniae saccharides 1, 4, 5, 6B, 7F,9V, 14, 18C, and 19F. In one embodiment the further antigens comprise S.pneumoniae saccharides 1, 4, 5, 7F, 9V, 14, 18C, 19F and 23F. In oneembodiment the further antigens comprise S. pneumoniae saccharides 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. In one embodiment thefurther antigens comprise S. pneumoniae saccharides 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A and 19F. In one embodiment the further antigenscomprise S. pneumoniae saccharides 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A,19F and 23F. In one embodiment the further antigens comprise S.pneumoniae saccharides 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and23F. In one embodiment the further antigens comprise S. pneumoniaesaccharides 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A and 19F. In oneembodiment the further antigens comprise S. pneumoniae saccharides 1, 4,5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F.

Any of the saccharides listed as ‘further antigens’ are optionallyconjugated to a carrier protein either by the process of the inventionor by a different process. Optionally these further antigens areconjugated to the carrier proteins listed above.

In an embodiment, the further antigens comprise S. pneumoniae capsularsaccharide 1 conjugated to protein D or CRM197. In an embodiment, thefurther antigens comprise S. pneumoniae capsular saccharide 3 conjugatedto protein D, CRM197, pneumolysin or PhtD or fragment or fusion proteinthereof. In an embodiment, the further antigens comprise S. pneumoniaecapsular saccharide 4 conjugated to protein D or CRM197. In anembodiment, the further antigens comprise S. pneumoniae capsularsaccharide 5 conjugated to protein D or CRM197. In an embodiment, thefurther antigens comprise S. pneumoniae capsular saccharide 6Bconjugated to protein D or CRM197. In an embodiment, the furtherantigens comprise S. pneumoniae capsular saccharide 7F conjugated toprotein D or CRM197. In an embodiment, the further antigens comprise S.pneumoniae capsular saccharide 9V conjugated to protein D or CRM197. Inan embodiment, the further antigens comprise S. pneumoniae capsularsaccharide 14 conjugated to protein D or CRM197. In an embodiment, thefurther antigens comprise S. pneumoniae capsular saccharide 23Fconjugated to protein D or CRM197. In an embodiment, the furtherantigens comprise S. pneumoniae capsular saccharide 18C conjugated totetanus toxoid or CRM197. In an embodiment, the further antigenscomprise S. pneumoniae capsular saccharide 19A conjugated to pneumolysinor CRM197. In an embodiment, the further antigens comprise S. pneumoniaecapsular saccharide 22F conjugated to CRM197 or PhtD or fragment offusion protein thereof. In an embodiment, the further antigens compriseS. pneumoniae capsular saccharide 6A conjugated to pneumolysin or a H.influenzae protein, optionally protein D or PhtD or fusion proteinthereof or CRM197. In an embodiment, the further antigens comprise S.pneumoniae capsular saccharide 6C conjugated to pneumolysin or a H.influenzae protein, optionally protein D or PhtD or fusion proteinthereof or CRM197. In an embodiment, the further antigens comprise S.pneumoniae capsular saccharide 19F conjugated to Diphtheria toxoid (DT).

The further antigens may also comprise Streptococcus pneumoniaeproteins. In one embodiment the further antigens comprise at least 1protein selected from the group consisting of the Poly Histidine Triadfamily (PhtX), Choline Binding protein family (CbpX), CbpX truncates,LytX family, LytX truncates, CbpX truncate-LytX truncate chimericproteins (or fusions), pneumolysin (Ply), PspA, PsaA, Sp128, Sp101,Sp130, Sp125 and Sp133.

The further antigens may also comprise antigens from further bacterialspecies. In one embodiment the vaccine or immunogenic compositioncomprises antigens originating from S. pneumoniae (S. pneumoniae),Haemophilus influenzae (H. Influenzae), Neisseria meningitidis (N.Meningitidis), Escherichia coli (E. col)i, Moraxella cattharlis (M.cattarhalis), tetanus, diphtheria, pertussis, Staphylococcus epidermidis(S. epidermidis), enterococci, Pseudomonas or Staphylococcus aureus (S.aureus).

In one embodiment the further antigens comprise M. cattarhalis antigens,preferred M. cattarhalis antigens are: OMP106 [WO 97/41731 (Antex) & WO96/34960 (PMC)]; OMP21; LbpA & LbpB [WO 98/55606 (PMC)]; TbpA & TbpB [WO97/13785 & WO 97/32980 (PMC)]; CopB [Helminen M E, et al. (1993) Infect.Immun. 61:2003-2010]; UspA1/2 [WO 93/03761 (University of Texas)]; andOmpCD. Examples of non-typeable Haemophilus influenzae antigens whichcan be included in a combination vaccine (especially for the preventionof otitis media) include: Fimbrin protein [(U.S. Pat. No. 5,766,608—OhioState Research Foundation)] and fusions comprising peptides therefrom[eg LB1(f) peptide fusions; U.S. Pat. No. 5,843,464 (OSU) or WO99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (StateUniversity of New York)]; TbpA and TbpB; Hia; Hmw-1,2; Hap; and D15.

In a further embodiment the further antigens comprise Diphtheria toxoid(DT), tetanus toxoid (TT), and pertussis components [typicallydetoxified Pertussis toxoid (PT) and filamentous haemagglutinin (FHA)with optional pertactin (PRN) and/or agglutinin 1+2], for example themarketed vaccine INFANRIX-DTPaTM (SmithKlineBeecham Biologicals) whichcontains DT, TT, PT, FHA and PRN antigens, or with a whole cellpertussis component for example as marketed by SmithKlineBeechamBiologicals s.a., as TritanrixTM. In a further embodiment the furtherantigens comprise Hepatitis B surface antigen (HepB).

In a further embodiment the further antigens comprise the PRP capsularsaccharide of H. influenzae (Hib).

In a further embodiment the further antigens comprise at least onecapsular saccharide from N. meningitidis A, C, W or Y. In a furtherembodiment the further antigens comprise at least one conjugate of acapsular saccharide from N. meningitidis A, C, W or Y.

The conjugate may also be mixed with an adjuvant. Suitable adjuvantsinclude, but are not limited to, aluminium salts (aluminium phosphate oraluminium hydroxide), monophosphoryl lipid A (for example 3D-MPL),saponins (for example QS21), oil in water emulsions, blebs or outermembrane vesicle preparations from Gram negative bacterial strains (suchas those taught by WO02/09746), lipid A or derivatives thereof, alkylglucosamide phosphates or combinations of two or more of theseadjuvants.

In a further embodiment the conjugate of the invention is mixed with apharmaceutically acceptable excipient.

In a further aspect of the invention there is provided a conjugateobtainable by the process of the invention. In a further aspect of theinvention there is provided a conjugate obtained by the process of theinvention. The invention also provides an immunogenic compositioncomprising the conjugate of the invention and a pharmaceuticallyacceptable excipient. In one embodiment the pharmaceutical acceptableexcipient does not contain a chloride salt, in a further embodiment thepharmaceutical excipient does not contain sodium chloride. In oneembodiment the pharmaceutical excipient comprises a buffer selected fromthe group consisting of maleate, tris, or citrate. In a furtherembodiment the buffer is maleate buffer.

The immunogenic composition of the invention may comprise furtherantigens, in particular those described as ‘further antigens’ above. Theimmunogenic composition may comprise an adjuvant, particularly thosedescribed above.

The invention also provides a vaccine comprising the immunogeniccomposition of the invention.

The vaccine preparations containing immunogenic compositions of thepresent invention may be used to protect or treat a mammal susceptibleto infection, by means of administering said vaccine via systemic ormucosal route. These administrations may include injection via theintramuscular, intraperitoneal, intradermal or subcutaneous routes; orvia mucosal administration to the oral/alimentary, respiratory,genitourinary tracts. Intranasal administration of vaccines for thetreatment of pneumonia or otitis media is possible (as nasopharyngealcarriage of pneumococci can be more effectively prevented, thusattenuating infection at its earliest stage). Although the vaccine ofthe invention may be administered as a single dose, components thereofmay also be co-administered together at the same time or at differenttimes (for instance pneumococcal saccharide conjugates could beadministered separately, at the same time or 1-2 weeks after theadministration of the any bacterial protein component of the vaccine foroptimal coordination of the immune responses with respect to eachother). In addition to a single route of administration, 2 differentroutes of administration may be used. For example, saccharides orsaccharide conjugates may be administered IM (or ID) and bacterialproteins may be administered IN (or ID). In addition, the vaccines ofthe invention may be administered IM for priming doses and IN forbooster doses.

The content of protein antigens in the vaccine will typically be in therange 1-100 μg, optionally 5-50 μg, most typically in the range 5-25 μg.Following an initial vaccination, subjects may receive one or severalbooster immunizations adequately spaced.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

Although the vaccines of the present invention may be administered byany route, administration of the described vaccines into the skin (ID)forms one embodiment of the present invention. Human skin comprises anouter “horny” cuticle, called the stratum corneum, which overlays theepidermis. Underneath this epidermis is a layer called the dermis, whichin turn overlays the subcutaneous tissue. Researchers have shown thatinjection of a vaccine into the skin, and in particular the dermis,stimulates an immune response, which may also be associated with anumber of additional advantages. Intradermal vaccination with thevaccines described herein forms an optional feature of the presentinvention.

The conventional technique of intradermal injection, the “mantouxprocedure”, comprises steps of cleaning the skin, and then stretchingwith one hand, and with the bevel of a narrow gauge needle (26-31 gauge)facing upwards the needle is inserted at an angle of between 10-15°.Once the bevel of the needle is inserted, the barrel of the needle islowered and further advanced whilst providing a slight pressure toelevate it under the skin. The liquid is then injected very slowlythereby forming a bleb or bump on the skin surface, followed by slowwithdrawal of the needle.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO 99/34850 and EP 1092444, also the jetinjection devices described for example in WO 01/13977; U.S. Pat. No.5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat.No. 5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S.Pat. No. 5,704,911, U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,893,397,U.S. Pat. No. 5,466,220, U.S. Pat. No. 5,339,163, U.S. Pat. No.5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat.No. 5,520,639, U.S. Pat. No. 4,596,556, U.S. Pat. No. 4,790,824, U.S.Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO 97/37705 and WO97/13537. Alternative methods of intradermal administration of thevaccine preparations may include conventional syringes and needles, ordevices designed for ballistic delivery of solid vaccines (WO 99/27961),or transdermal patches (WO 97/48440; WO 98/28037); or applied to thesurface of the skin (transdermal or transcutaneous delivery WO 98/20734;WO 98/28037).

When the vaccines of the present invention are to be administered to theskin, or more specifically into the dermis, the vaccine is in a lowliquid volume, particularly a volume of between about 0.05 ml and 0.2ml.

The content of antigens in the skin or intradermal vaccines of thepresent invention may be similar to conventional doses as found inintramuscular vaccines (see above). However, it is a feature of skin orintradermal vaccines that the formulations may be “low dose”.Accordingly the protein antigens in “low dose” vaccines are optionallypresent in as little as 0.1 to 10 μg or 0.1 to 5 μg per dose; and thesaccharide (optionally conjugated) antigens may be present in the rangeof 0.01-1 μg, or between 0.01 to 0.5 μg of saccharide per dose.

As used herein, the term “intradermal delivery” means delivery of thevaccine to the region of the dermis in the skin. However, the vaccinewill not necessarily be located exclusively in the dermis. The dermis isthe layer in the skin located between about 1.0 and about 2.0 mm fromthe surface in human skin, but there is a certain amount of variationbetween individuals and in different parts of the body. In general, itcan be expected to reach the dermis by going 1.5 mm below the surface ofthe skin. The dermis is located between the stratum corneum and theepidermis at the surface and the subcutaneous layer below. Depending onthe mode of delivery, the vaccine may ultimately be located solely orprimarily within the dermis, or it may ultimately be distributed withinthe epidermis and the dermis.

In one aspect of the invention is provided a vaccine kit, comprising avial containing an immunogenic composition of the invention, optionallyin lyophilised form, and further comprising a vial containing anadjuvant as described herein. It is envisioned that in this aspect ofthe invention, the adjuvant will be used to reconstitute the lyophilisedimmunogenic composition.

A further aspect of the invention is a method of immunising a human hostagainst bacterial disease infection comprising administering to the hostan immunoprotective dose of the immunogenic composition or vaccine orkit of the invention. A further aspect of the invention is a method ofimmunising a human host against infection caused by S. pneumoniae and/orHaemophilus influenzae comprising administering to the host animmunoprotective dose of the immunogenic composition or vaccine or kitof the invention.

A further aspect of the invention is an immunogenic composition of theinvention for use in the treatment or prevention of bacterial disease. Afurther aspect of the invention is an immunogenic composition of theinvention for use in the treatment or prevention of disease caused by S.pneumoniae and/or Haemophilus influenzae infection

A further aspect of the invention is use of the immunogenic compositionor vaccine or kit of the invention in the manufacture of a medicamentfor the treatment or prevention of bacterial diseases. A further aspectof the invention is use of the immunogenic composition or vaccine or kitof the invention in the manufacture of a medicament for the treatment orprevention of diseases caused by S. pneumoniae and/or Haemophilusinfluenzae infection.

The invention also provides an activated bacterial saccharide, whereinthe activated bacterial saccharide comprises a repeat unit of formula(I):

wherein the activated bacterial saccharide comprises n repeat units andn is between 2 and 2400, between 20 and 2000, between 50 and 1500,between 1000 and 2000, between 1000 and 2500 or between 1500 and 2300.wherein at least 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 5%, 10% or 30% butless than 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 30% or 50% of S1is

and the remainder is

wherein S2 is either

and wherein S3 is either

In an embodiment less than 0.001%, 0.1%, 0.5% 1%, 2%, 3%, 5%, 10%, 30%or 50% of S2 is

In an embodiment less than 0.1%, 0.5%, 1%, 2%, 3%, 5%, 10%, 30% or 50%of S3 is

“Around” or “approximately” are defined as within 10% more or less ofthe given figure for the purposes of the invention.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

Embodiments herein relating to “vaccine compositions” of the inventionare also applicable to embodiments relating to “immunogeniccompositions” of the invention, and vice versa.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES Example 1 Oxidation of 23F and 6B Using Periodate

Polysaccharides (PS) 23F or 6B were dissolved in 100 mM KH₂PO₄ (pH 7.4),10 mM KH₂PO₄ or WFI, to form solutions of 2 mg PS/ml. The solution wasincubated for 2 hours under agitation at room temperature. After thistime the pH was adjusted to pH 6.0 with 1 NHCl. Periodate was added as apowder or in liquid form (10 mg/ml in WFI) in various amounts to achievea range of molar ratios (table 1). The solutions were incubated for 17hours at room temperature (20-25° C.), after which time the samples weredialyzed or diafiltered against WFI.

High performance gel filtration chromatography coupled with refractiveindex and multiangle laser lights scattering (MALLS-) detectors was usedto measure the molecular weight. Size exclusion media(TSK5000PWXL-Tosoh) was used to profile the molecular size distributionof the polysaccharide (elution 0.5 ml/min in NaCl 0.2M-NaN3 0.02%).

Table 1 and FIG. 1 describe the results of these experiments. Thesedemonstrate that for the 23F saccharide substantial sizing occurs onoxidation using high molar equivalents of periodate in 100 mM phosphatebuffer. This sizing effect can be reduced by reducing the concentrationof phosphate buffer or the molar equivalents of periodate used.

TABLE 1 23F 6B molar molar equivalent equivalent of Size Sam- of SizeSample periodate Buffer (KDa) ple periodate buffer (KDa) 23F 0 Water 8616B 0 10 mM 102 native phos- phate 23F 0 10 mM 847 6B 0.1 10 mM 97 nativephosphate phos- phate 23F 0 100 mM 860 6B 0.2 10 mM 99 native phosphatephos- phate 23F 0 100 mM 1655 6B 0.3 10 mM 96 ATCC phosphate phos-native phate 23F 1 100 mM <1 6B 0.75 10 mM 86 phosphate phos- phate 23F1 Water 36 23F 1.2 100 mM <1 phosphate 23- 1 100 mM 2 FATCC phosphate23- 0.125 100 mM 39 FATCC phosphate 23F 0.1 10 mM 466.9 phosphate 23F0.15 10 mM 398.5 phosphate 23F 0.2 10 mM 336 phosphate 23F 0.5 10 mM179.1 phosphate

Example 2 Conjugation of 23F to CRM197 Using Reductive Amination andCDAP Chemistry Reductive Amination

1 g of PS23F was dissolved in 500 ml of 10 mM KH₂PO₄, pH 7.15. Thissolution was incubated at room temperature for two hours. The pH wasadjusted to 6.0N with 1M HCl. 111 mg of periodate (NaIO₄, 0.4 molarequivalents of periodate) was added to the PS23F solution, and thesolution was incubated for 17 hours in the dark at room temperature tooxidise PS23F. The solution was then diafiltered against WFI.

The activated PS23F was lyophilised with the CRM197 protein (at a CRM/PSratio (w/w): 0.625) in the presence of a stabilising agent.

900 mg of the lyophilised PS23F/CRM197 mixture was solubilised byaddition of 350 ml of DMSO solvent and incubating for 2 hours at roomtemperature. To reduce the PS23F/CRM197 mixture 1 molar equivalent ofNaBH₃CN was added (735 μl of a solution of 100 mg/ml in WFI). Thesolution was incubated for a further 40 hours room temperature (15°C.-25° C.) under agitation. After this time 2 molar equivalent of NaBH₄(100 mg/ml in WFI) was added and the solution incubated for 4 hours atroom temperature. 2200 ml of 150 mM NaCl was added before diafiltration(cut-off 100 kDa) and purification by DEAE. The fractions of interestwere pooled and filtered through a 0.22 μm filter.

CDAP

200 mg of microfluidized PS23F was dissolved in water until aconcentration of 10 mg/ml was obtained. NaCl was added to this solutionat a final concentration of 2M.

Sufficient CDAP solution (100 mg/ml freshly prepared in 5/50 v/vacetonitrile/WFI) was added to reach a CDAP:PS ratio of 0.75 mg/mg PS.

After 90 seconds, the pH was raised to pH 9.5 by addition of 0.1 N NaOH.

3 minutes later sufficient CRM197 (10 mg/ml in 0.15M NaCl) was added toreach a ratio of 1.5 (CRM197:PS (w/w)), the pH was maintained at pH 9.5.This solution was incubated for 1 hour at pH 9.5.

After this coupling step, 10 ml of 2M glycine solution was added to themixture and the pH was adjusted to pH9.0 (the quenching pH). Thesolution was stirred for 30 minutes at room temperature. The conjugatewas purified using a 5 μm filter followed by Sephacryl S400HR (XK50/100)which removes small molecules and unconjugated polysaccharides andprotein. The flow rate was fixed at 150 ml/hour. Elution was achievedusing 150 mM NaCl. The fractions of interest were pooled and filteredusing Milipack 20. The resulting conjugate had a final CRM197/PS ratio(w/w) of 1.35/w.

Example 3 Immunogenicity of 23F-CRM197 Conjugates Made by ReductiveAmination and CDAP Chemistry

Conjugates were made using the methods described in example 2. Femaleguinea pigs were immunized intramuscularly three times (at days 0, 14and 28) with 0.25 μg of the PS23F-CRM197 conjugates. Animals were bledon day 42 and the antibody response directed against PS23F was measuredby ELISA and OPA.

ELISA

Microplates were coated with purified pneumococcal polysaccharide in PBSbuffer. The plates were washed four times with 0.9% NaCl and 0.05% Tween20. Sera were incubated for 1 hour at 37° C. with CPS (V/V) in PBS 0.05%Tween 20. Sera were added to the microwells and serially diluted(two-fold dilution step) in PBS-0.05% Tween. The plates were incubatedunder agitation for 30 minutes at room temperature. The plates werewashed as above and an anti-guinea pig IgG antibodies peroxydaseconjugate was added, the plates were then incubated for 30 minutes atRT. After washing, the substrate (4 mg of OPDA in 10 ml of citrate 0.1MpH 4.5 and 5 μl of H₂O₂) was added to each well for 15 minutes. Thereaction was stopped by addition of HCl 1N. Absorbance was read at490-620 nm using a spectrophotometer. The colour developed is directlyproportional to the amount of antibody present in the serum. The levelof anti-PS IgG present in the sera is determined by comparison to thereference curve serum added on each plate and expressed in μg/ml.

Results were analysed statistically after assuming homogeneity ofvariance (checked by Cochrans's C test) and normality (checked using theShapiro-Wilk test). All statistics were carried out using Anova(Tukey-HSD) on log transformation concentration IgG.

Opsonophagocytosis

Serum samples were heated for 45 min at 56° C. to inactivate anyremaining endogenous complement. Twenty-five microlitre aliquots of each1:2 diluted serum sample were serially diluted (two fold) in 25 μl OPAbuffer (HBSS—14.4% inactivated FBS) per well of a 96-well round bottommicrotitre plate. Subsequently, 25 μl of a mixture of activated HL-60cells (1×107 cells/ml), freshly thawed pneumococcal working seed andfreshly thawed baby rabbit complement in an e.g. 4/2/1 ratio (v/v/v) wasadded to the diluted sera to yield a final volume of 50 μl. The assayplate was incubated for 2 h at 37° C. with orbital shaking (210 rpm) topromote the phagocytic process. The reaction was stopped by laying themicroplate on ice for at least 1 min. A 20 μl aliquot of each well ofthe plate was then transferred into the corresponding well of a 96-wellflat bottom microplate and 50 μl of Todd-Hewitt Broth-0.9% agar wasadded to each well. After overnight incubation at 37° C. and 5% CO2,pneumococcal colonies appearing in the agar were counted using anautomated image analysis system (KS 400, Zeiss, Oberkochen, Germany).Eight wells without serum sample were used as bacterial controls todetermine the number of pneumococci per well. The mean number of CFU ofthe control wells was determined and used for the calculation of thekilling activity for each serum sample. The OPA titre for the serumsamples was determined by the reciprocal dilution of serum able tofacilitate 50% killing of the pneumococci. The opsonophagocytic titrewas calculated by using a 4-parameter curve fit analysis.

Results were analysed statistically after assuming homogeneity ofvariance (checked by Cochrans's C test) and normality (checked using theShapiro-Wilk test). All statistics were performed by Anova (Tukey-HSD)on log transformation concentration IgG for ELISA and Kruskal-Wallis onlog dilution for OPA.

A significantly higher antibody response was induced in the guinea pigsafter immunisation with PS23F-CRM197 conjugated by reductive aminationthan PS23F-CRM197 conjugated by CDAP chemistry as seen in FIG. 2.

TABLE 2 23F-CRM197 made by 23F-CRM197 made by Assay reductive aminationCDAP ELISA liter (μ/ml) 213.3 40.5 OPA (50% killing) 9232 591

Example 4 A Further Example of Reductive Amination of 23F 23F-CRM-RA-116

150 mg of native PS23F (PS23FP114) was dissolved at a concentration of 2mg/ml in 10 mM phosphate buffer (pH 7.2) for 4 hours. After dissolution,pH was adjusted to pH 6.0 with 1N HCl. Then 0.4 molar equivalent ofperiodate (Na IO₄) was added to the PS solution and incubated for 17 hrsin the dark at 25° C. The solution is then diafiltered (cut off 30 kDa)against WFI and the oxidised PS was filtered on 0.22 μm membrane.

50 mg of oxidised PS and 75 mg of CRM197 were lyophilized together(CRM/PS ratio (w/w): 1.5/1) in the presence of a stabilising agent.Lyophilized PS+CRM197 was solubilised with 20 ml of DMSO for 2 hrs atroom temperature (15-25° C.). 1 molar equivalent of TAB (Sodiumtriacetoxyborohydride) was then added (13.7 mg) and after 17 hrs underagitation, 2 molar equivalent of NaBH₄ (100 mg/ml in 0.1M NaOH) wasadded followed by an incubation at room temperature for 30 minutes. Thesolution was diluted 5× by addition of WFI followed by a diafiltration(cut-off 30 kDa) against 10 mM phosphate buffer, 150 mM NaCl pH 7.2. Theconjugate was then loaded onto DEAE resin and eluted in 10 mM phosphatebuffer, 500 mM NaCl pH 7.2. The conjugate was finally filtered on 0.22μm. The resulting conjugate has a final CRM/PS ratio (w/w) of 2.3/1.

For further conjugates, a second diafiltration step was added after DEAEcolumn in order to change the buffer (150 mM NaCl as final buffer).

Example 5 Conjugation of 6B to CRM197 Using Reductive Amination (withDifferent Protein:Saccharide Ratios and Different Sized Microfluidised6B Saccharides) and CDAP Chemistry 6B-CRM-RA-122

200 mg of microfluidized PS6B (84 kDa, 11.7 mg/ml) was diluted at 2mg/ml in 10 mM phosphate buffer (pH 7.2). pH was adjusted to pH 6.0 with1N HCl. Then 0.1 molar equivalent of periodate (Na IO₄) was added to thePS solution and incubated for 17 hrs in the dark at room temperature.The solution is then diafiltered (cut off 30 kDa) against WFI. 50 mg ofPS and 30 mg of CRM197 were lyophilized together (CRM/PS ratio (w/w):0.6/1) in the presence of a stabilising agent. Lyophilized PS+CRM197were solubilised with 20 ml of DMSO for 3 hrs at room temperature. Then2.5 molar equivalent of TAB (Sodium triacetoxyborohydride) was added(38.7 mg) and after 16 hrs under agitation, 2 molar equivalent of NaBH₄(100 mg/ml in 0.1M NaOH) was added followed by an incubation at roomtemperature for 30 minutes. The solution was diluted 4× by addition ofWFI followed by a diafiltration (cut-off 100 kDa). The conjugate wasthen filtered on 0.22 μm. The resulting conjugate has a final CRM/PSratio (w/w) of 1.1/1.

6B-CRM-RA-123:

Microfluidized PS6B (84 kDa) was conjugated to CRM197 as described for6B-CRM-RA-122 except the freeze-drying step was carried out using aninitial CRM197/PS ratio (w/w) of 2/1 and 30 ml of DMSO was used for thedissolution in DMSO step (instead of 20 ml). The resulting conjugate hada final CRM/PS ratio (w/w) of 3.0/1.

6B-CRM-RA-124:

200 mg of microfluidized PS6B (350 kDa, 11.7 mg/ml) having a molecularweight of 350 kDa was diluted to 2 mg/ml in 10 mM phosphate buffer (pH7.2). pH was adjusted to pH 6.0 with 1N HCl. Then 0.1 molar equivalentof periodate (Na IO₄) was added to the PS solution and incubated for 17hrs in the dark at room temperature. The solution is then diafiltered(cut off 100 kDa) against WFI. 50 mg of PS and 60 mg of CRM197 werelyophilized together (CRM/PS ratio (w/w): 1.2/1) in the presence of astabilising agent. Lyophilized PS+CRM197 were solubilised with 20 ml ofDMSO for 5 hrs at room temperature. 2.5 molar equivalent of TAB (Sodiumtriacetoxyborohydride) was then added (38.7 mg) and after 16 hrs underagitation, 2 molar equivalent of NaBH4 (100 mg/ml in 0.1M NaOH) wereadded followed by incubation for 30 min at room temperature. Thesolution was diluted 4× by addition of WFI followed by a diafiltration(cut-off 100 kDa). The conjugate was then filtered on 0.22 μm. Theresulting conjugate has a final CRM/PS ratio (w/w) of 1.6/1.

6B-CRM-RA-125:

Microfluidized PS6B (350 kDa) was conjugated to CRM197 as described for6B-CRM-RA-124 except the freeze-drying step was carried out using aninitial CRM197/PS ratio (w/w) of 2/1 and the dissolution in DMSO wascarried out using 33 ml (instead of 20 ml). The resulting conjugate hada final CRM/PS ratio (w/w) of 2.9/1.

6B-CRM-003:

50 mg of microfluidized PS6B were diluted at 10 mg/ml in water (10mg/ml). NaCl in solid form was added to reach a final concentration of2M. CDAP solution (100 mg/ml freshly prepared in 50/50 v/vacetonitrile/WFI) was added to reach the appropriate CDAP/PS ratio (1.5mg/mg PS). After 1.5 minutes, the pH was raised to the activation pH 9.5by addition of 0.1N NaOH and was stabilised at this pH until addition ofCRM197. After 3 minutes, CRM197 (10 mg/ml in 0.15 M NaCl) was added toreach a ratio CRM197/PS (w/w) of 2; the pH was maintained at thecoupling pH 9.5. The solution was left for 2 hrs under pH regulation.

After the coupling step, 2.5 ml of 2M glycine solution was added to themixture. The pH was adjusted to the quenching pH (pH 9.0). The solutionwas stirred for 30 min at room temperature. Then the conjugate wasfiltered using a 5 μm filter and injected on Sephacryl S400HR (XK26/100)column to remove small molecules (including DMAP) and unconjugated PSand protein. Flow rate was fixed at 30 ml/h. Elution was carried out in150 mM NaCl. Interesting fractions were pooled and filtered on Millipack20. The resulting conjugate had a final CRM197/PS ratio (w/w) of 1.5/1.

6B-CRM-RA-144

1 g of microfluidized PS6B (245 kDa, 9.47 mg/ml) was diluted to 2 mg/mlin 10 mM phosphate buffer (pH 7.2). The pH was adjusted to pH 6.0 with1N HCl. 0.1 molar equivalent of periodate (NaIO₄) was then added to thePS solution and incubated for 18 hrs in the dark at room temperature.The solution was then diafiltered against WFI (Sartocon Slice200Hydrosart 100 kDa). 200 mg of oxidized PS and 240 mg of CRM197 werelyophilized together (CRM/PS ratio (w/w): 1.2/1) in the presence of astabilising agent. Lyophilized PS+CRM197 were solubilised with 80 ml ofDMSO for 6 hrs at 25° C. Then 2.5 molar equivalent of TAB (Sodiumtriacetoxyborohydride) was added (154.9 mg) and after 16 hrs underagitation at 25° C., 2 molar equivalent of NaBH₄(100 mg/ml in 0.1M NaOH)was added and incubated for 30 min. The solution was diluted 5× in WFIand after 30 min was diafiltered 10× with 150 mM NaCl and then 5× withPO₄ (K/K₂) 10 mM pH7.2+150 mM NaCl (Sartorius Sartocon Slice 200Hydrosart 100 kDa). Then the retentate was loaded onto a DEAE column(XK26/40). The column was washed with PO₄ (K/K₂) 10 mM pH7.2/NaCl 150 mMbuffer. The conjugate was eluted with PO₄ (K/K₂) 10 mM pH7.2/NaCl 500 mMbuffer. The eluate was concentrated and diafiltered with 5 volumes of150 mM NaCl and then filtered on 0.22 μm filter. The resulting conjugatehas a final CRM/PS ratio (w/w) of 1.6/1.

Example 6 Immunogenicity of 6B-CRM197 Conjugates Made by ReductiveAmination and CDAP Chemistry

Groups of 40 female Balb/c mice (4 weeks-old) were immunizedintramuscularly three times at days 0, 14 and 28 with 0.1 μg of PS6Bconjugates produced by reductive amination or CDAP chemistry formulatedon AlPO₄. PS6B-PD was used as benchmark. Mice were bled on day 42 andthe antibody response directed against each antigen was measured byELISA and OPA.

Groups of 20 female guinea pig (150 gr from Hartley) were immunizedintramuscularly three times at days 0, 14 and 28 with 0.25 μg of PS6Bconjugates produced by reductive amination or CDAP chemistry adjuvantedwith AlPO₄. PS6B-PD was used as benchmark. Guinea pigs were bled on day42 and the antibody response directed against each antigen was measuredby ELISA and OPA.

Mouse and Guinea Pig OPA

Serum samples were heated for 45 min at 56° C. to inactivate anyremaining endogenous complement. Twenty-five microlitre aliquots of each1:2 diluted serum sample was two-fold serially diluted in 25 μl OPAbuffer (HBSS—14.4% inactivated FBS) per well of a 96-well round bottommicrotitre plate. Subsequently, 25 μl of a mixture of activated HL-60cells (1×10⁷ cells/ml), freshly thawed pneumococcal working seed andfreshly thawed baby rabbit complement in an e.g. 4/2/1 ratio (v/v/v)were added to the diluted sera to yield a final volume of 50 μl. Theassay plate was incubated for 2 h at 37° C. with orbital shaking (210rpm) to promote the phagocytic process. The reaction was stopped bylaying the microplate on ice for at least 1 min. A 20 μl aliquot of eachwell of the plate was then transferred into the corresponding well of a96-well flat bottom microplate and 50 μl of Todd-Hewitt Broth-0.9% agarwas added to each well. After overnight incubation at 37° C. and 5% CO₂,pneumococcal colonies appearing in the agar were counted using anautomated image analysis system (KS 400, Zeiss, Oberkochen, Germany).Eight wells without serum sample were used as bacterial controls todetermine the number of pneumococci per well. The mean number of CFU ofthe control wells was determined and used for the calculation of thekilling activity for each serum sample. The OPA titre for the serumsamples was determined by the reciprocal dilution of serum able tofacilitate 50% killing of the pneumococci. The opsonophagocytic titrewas calculated by using a 4-parameter curve fit analysis.

Table 3 Describes the GMC Levels Obtained by Immunisation of Balb/C Micewith the Conjugates Made Using the Methods of Example 4.

TABLE 3 G1 G2 G3 G4 G5 G6 Subject/Result PS06B-CRM122 PS06B-CRM123PS06B-CRM124 PS06B-CRM125 PS06B-CRM003 PS06B-PD (R: 1/1, PS 84 (R: 3/1,PS 84 kDa) (R: 1.5/1, PS 350 kDa) (R: 2.9/1, PS 350 kDa) (CDAP) GMC(UG-ML) 0.83 0.37 1.18 0.64 0.31 0.10 Responders (%) 31/40 26/40 33/4029/40 29/40 15/40

The immunogenicity of these conjugates in balb/c mice is described inFIG. 3. Together FIG. 3, and table 3 demonstrate that in the mouse modelthe conjugates produced by reductive amination were comparable withthose produced using CDAP chemistry. In particular FIG. 3 demonstratesthat the immunogenicities of the conjugates produced using reductiveamination was higher than the immunogenicity of the conjugate made usingCDAP chemistry.

Table 4 describes the GMC levels obtained by immunisation of guinea pigswith the conjugates made using the methods of example 4.

TABLE 4 G1 G2 G3 G4 G5 G6 Subject/Result PS06B-CRM 122 PS06B-CRM123PS06B-CRM124 PS06B-CRM125 PS06B-CRM003 (CDAP) PS06B-PD (R: 1/1, PS 84kDa) (R: 3/1, PS84 kDa) (R: 1.5/1, PS0350 kDa) (R: 2.9/1, PS 350 kDa)GMC (UG-ML) 3.51 7.70 2.84 19.93 3.70 1.55 Responders (%) 20/20 20/2020/20 20/20 20/20 20/20

The immunogenicity of these conjugates in guinea pigs is described inFIG. 4. Similar to the experiments carried out in the mouse model, theresults in table 4 and FIG. 4 show that the conjugates produced byreductive amination were comparable with those produced using CDAPchemistry, in particular PS06B-CRM125 demonstrated significantly higherGMC levels and immunogenicities than the conjugate produced using CDAP.

Example 7 Conjugation of Hib to Tetanus Toxoid Using Reductive AminationHib-IO4-LS080

2.9 g of PS (orcinol dosage, AHIBCPA007 lot) were dissolved in 260 ml of10 mM phosphate buffer (Na/K₂) pH 6.2 for 4 h30 at room temperature andthen overnight at +4° C. The viscosity follow-up was done during thedissolution. After 4 hours dissolution the viscosity seemed to bestable. PS was diluted at 10 mg/ml with phosphate buffer and thenoxidised in the dark with 0.07 molar equivalent of NaIO₄ during 60minutes. Oxidised PS was diafiltered (Sartorius Hydrosart 2 kDa) against3.5 volumes of phosphate buffer and then filtered on a 0.22 μm filter.The number of repeating units obtained after oxidation was estimated by¹H-NMR and was found to be around 21.

Hib-TT-LS210, 212 and 213

200 mg of oxidised PS (14.56 mg/ml) were mixed with 300 mg of TT (31.18mg/ml, TT/PS ratio (w/w): 1.5/1) and diluted to 4 mg/ml with 36.64 ml of10 mM phosphate buffer (Na/K₂) pH 6.2. The solution was lyophilized inthe presence of a stabilising agent. Lyophilized PS+TT was solubilisedwith 20 ml of DMSO for 6 hrs at 25° C. Then 10 Meq of TAB (Sodiumtriacetoxyborohydride) were added (38.7 mg) and after 16 hrs underagitation, 2 molar equivalent of NaBH₄ (100 mg/ml in 0.1M NaOH) wasadded followed by an incubation for 30 min at room temperature. Thesolution was diluted 3× by addition of WFI followed by a diafiltrationstep (5 volumes of WFI followed by 5 volumes of 10 mM acetate buffer 150mM NaCl pH 6.2, 100 kDa MWCO). The sample was then loaded on SephacrylS300HR resin. Elution was carried out in 10 mM acetate buffer using 150mM NaCl (pH 6.2). Interesting fractions were pooled and filtered on a0.22 μm filter. The resulting conjugates had a final TT/PS ratio (w/w)of 2.1/1.

1. A process for conjugating a bacterial saccharide comprising the stepsof a) reacting the bacterial saccharide with 0.001-0.7, 0.005-0.5,0.01-0.5, 0.1-1.2, 0.1-0.5, 0.1-0.2, 0.5-0.8, 0.1-0.8, 0.3-1.0 or0.4-0.9 molar equivalents of periodate to form an activated bacterialsaccharide; b) mixing the activated bacterial saccharide with a carrierprotein; c) reacting the activated bacterial saccharide and the carrierprotein with a reducing agent to form a conjugate; or a) reacting thebacterial saccharide with 0.001-0.7, 0.005-0.5, 0.01-0.5, 0.1-1.2,0.1-0.5, 0.1-0.2, 0.5-0.8, 0.1-0.8, 0.3-1.0 or 0.4-0.9 molar equivalentsof periodate to form an activated bacterial saccharide; b) mixing theactivated bacterial saccharide with a linker; c′) reacting the activatedbacterial saccharide with the linker using a reducing agent to form abacterial saccharide-linker; d) reacting the bacterial saccharide-linkerwith a carrier protein to form a conjugate; wherein step a) occurs in abuffer which does not contain an amine group, and the buffer has aconcentration between 1-100 mM.
 2. The process of claim 1 wherein thebuffer is selected from the group consisting of phosphate buffer, boratebuffer, acetate buffer, carbonate buffer and citrate buffer.
 3. Theprocess of any one of claims 1-2 wherein the buffer is an inorganicbuffer.
 4. The process of any one of claims 1-3 wherein the buffer isphosphate buffer.
 5. The process of any one of claims 1-4 wherein thebuffer has a concentration between 1-50 mM, 1-25 mM, 1-10 mM, 5-15 mM,8-12 mM, or 10-50 mM or around 10 mM.
 6. The process of any one ofclaims 1-5 wherein the pH in step a) is pH 3.5-8.0 5.0-7.0, or pH5.5-6.5 or around pH 6.0.
 7. The process of any one of claims 1-6wherein the bacterial saccharide comprises a bacterial capsularsaccharide.
 8. The process of any one of claims 1-7 wherein thebacterial saccharide originates from S. pneumoniae, H. influenzae, N.meningitidis, S. aureus, enterococci, Salmonella Vi, or S. epidermidis.9. The process of any one of claims 1-8 wherein the bacterial saccharideis an S. pneumoniae capsular saccharide selected from the groupconsisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
 10. The process of anyone of claims 1-9 wherein the bacterial saccharide is selected from thegroup consisting of 5, 6B, 6A, 7F, 9V, 14, 19F or 23F.
 11. The processof any one of claims 1-10 wherein the bacterial saccharide is S.pneumoniae capsular saccharide 23F.
 12. The process of any one of claims1-10 wherein the bacterial saccharide is S. pneumoniae capsularsaccharide 6B.
 13. The process of any one of claims 1-11 wherein thebacterial saccharide is S. pneumoniae capsular saccharide 6A.
 14. Theprocess of any one of claims 1-8 wherein the bacterial saccharide isHaemophilus influenzae b (Hib) polysaccharide or oligosaccharide. 15.The process of any one of claims 1-14 wherein the bacterial saccharidecontains vicinal diols.
 16. The process of any one of claims 1-15wherein the bacterial saccharide is either a native polysaccharide or isreduced in size by a factor of no more than x20 (for instance bymicrofluidization).
 17. The process of any one of claims 1-16 whereinthe bacterial saccharide is microfluidised before step a).
 18. Theprocess of any one of claims 16-17 wherein the average molecular weightof the native polysaccharide is between 20 kDa and 2000 kDa or between30 kDa and 1000 kDa.
 19. The process of claim 18 wherein the averagemolecular weight of the native polysaccharide is between 750-1500 kDa.20. The process of claim 18 wherein the average molecular weight of thenative polysaccharide is between 100 and 250 kDa.
 21. The process of anyone of claims 1-20 wherein the average molecular weight of the bacterialsaccharide is between 1-1100 kDa, 100-470 kDa, 200-300 kDa, 600-1100 kDaor 800-1000 kDa after step a).
 22. The process of claim 11 wherein theaverage molecular weight of the 23F saccharide is between 100-470 kDa or200-300 kDa after step a).
 23. The process of claim 14 wherein theaverage molecular weight of the Hib bacterial saccharide is between 1and 50 kDa or between 5 and 10 kDa after step a).
 24. The process of anyone of claims 1-23 wherein the carrier protein is selected from thegroup consisting of tetanus toxoid, fragment C of tetanus toxoid,diphtheria toxoid, CRM197, Pneumolysin, protein D, PhtD, PhtDE and N19.25. The process of any one of claims 1-25 wherein step a) is carried outin the dark.
 26. The process of claim 15 wherein less than 0.001%,0.01%, 0.1%, 0.5%, 1%, 2%, or 5% of the vicinal diols of the bacterialsaccharide become oxidised during step a).
 27. The process of claim 26wherein during step c) the carbonyl group reacts with an amine group onthe carrier protein.
 28. The process of any one of claims 1-27 whereinthe bacterial saccharide is present at a concentration of between 0.2g/l and 14 g/l, 8 g/l and 12 g/l, 10 g/l and 12 g/l, 1 g/l and 4 g/l 0.2g/l and 1 g/l or between 0.4 g/l and 0.6 g/l in step a).
 29. The processof any one of claims 1-28 wherein the initial concentration of carrierprotein in step b) is between 0.5 g/l and 35 g/l, 25 g/l and 35 g/l, or0.5 g/l and 5 g/l.
 30. The process of any one of claims 1-29 wherein theinitial concentration of activated bacterial saccharide in step b) isbetween 0.2 g/l and 20 g/l, 10 g/l and 28 g/l, or between 0.2 g/l and 4g/l.
 31. The process of any one of claims 1-30 wherein the initial ratioof activated bacterial saccharide to carrier protein in step b) is 2.0:1to 0.1:1, 1.8:1 to 0.4:1, 1:1 to 1.4:1, 1.8:1 to 1.6:1, 1.4:1 to 1.6:1,0.8:1 to 0.4:1, 0.7:1 to 0.5:1, or 0.7:1 to 0.6:1 (wt/wt).
 32. Theprocess of any one of claims 1-31 wherein the final ratio of carrierprotein to bacterial saccharide after step c) or c′) is 0.5:1 to 4:1,0.8:1 to 3.2:1, 0.5:1 to 1.8:1, 1.4:1 to 1.8:1, 1:1 to 1.2:1, 2:1 to2.4:1, or 2.5:1 to 3.5:1.
 33. The process of any one of claims 1-32wherein the temperature of the reaction in step a) is maintained at4-40° C., 10-32° C., 17-30° C. or 22-27° C.
 34. The process of any oneof claims 1-33 wherein the temperature of the reaction in step c) ismaintained at 4-40° C., 10-32° C., 17-30° C. or 22-27° C.
 35. Theprocess of any one of claims 1-34 wherein step a) has a duration of lessthan 30 hours or between 15 and 25 hours, or 10 and 20 hours, between 30minutes and 25 hours, between 1 hour and 35 hours.
 36. The process ofany one of claims 1-35 wherein step c) has a duration of between 10-60,or 20-60 hours.
 37. The process of any one of claims 1-36 wherein theactivated bacterial saccharide is conjugated directly to the carrierprotein.
 38. The process of any one of claims 1-36 wherein the activatedbacterial saccharide is conjugated to the carrier protein through alinker.
 39. The process of claim 38 wherein the linker is between 1 and20 Angstroms in length.
 40. The process of any one of claim 38 or 39wherein the linker comprises an ADH linker.
 41. The process of any oneof claims 1-40 wherein the reducing agent comprises sodiumcyanoborohydride.
 42. The process of any one of claims 1-40 wherein thereducing agent comprises sodium triacetoxyborohydride.
 43. The processof claim 42 wherein between 0.5 and 2 or 0.8 and 1.2 molar equivalent ofreducing agent is used in step c).
 44. The process of claim 43 whereinbetween 2 and 10 molar equivalent of reducing agent is used in step c).45. The process of any one of claims 1-44 wherein any unreacted carbonylgroups are capped by reaction with sodium borohydride (NaBH₄).
 46. Theprocess of claim 45 wherein around 2M equivalents of sodium borohydrideare used.
 47. The process of any one of claim 45 or 46 wherein theproduct of step c) is reacted with sodium borohydride for 15 mins-15hrs, 15 mins-45 mins, 2-10 hrs, or 3-5 hrs.
 48. The process of any oneof claims 1-47 wherein the activated bacterial saccharide and thecarrier protein are lyophilised before step b).
 49. The process of anyone of claims 1-48 wherein the activated bacterial saccharide and thecarrier protein are lyophilised before step c).
 50. The process of anyone of claims 48-49 wherein the activated bacterial saccharide and thecarrier protein are lyophilised in the presence of a non-reducing sugar.51. The process of any one of claims 48-50 wherein the non-reducingsugar is selected from the group consisting of sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit.
 52. The process of any one of claims 1-51 steps b) and/or c)are carried out in DMSO solvent.
 53. The process of any one of claims1-51 wherein step b) and/or step c) are carried out in DMF solvent. 54.The process of any one of claims 1-53 comprising a further step e) ofpurifying the conjugate.
 55. The process of claim 54 wherein step e)comprises purifying the conjugate using diafiltration.
 56. The processof any one of claims 54-55 wherein step e) comprises purifying theconjugate using ion exchange chromatography.
 57. The process of claim 56wherein step e) comprises purifying the conjugate using size exclusionchromatography.
 58. The process of any one of claims 1-57 comprising afurther step f), wherein the conjugate is sterile filtered.
 59. Theprocess of any one of claims 1-58 containing a further step of mixingthe conjugate with further antigens.
 60. The process of claim 59 whereinthe further antigens comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 S. pneumoniae saccharides selected from the groupconsisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
 61. The process of anyone of claims 59-60 wherein the further antigens comprise S. pneumoniaesaccharides 4, 9V, 14, 18C, and 19F.
 62. The process of any one ofclaims 59-61 wherein the further antigens comprise S. pneumoniaesaccharide 19A.
 63. The process of any one of claims 59-62 wherein thefurther antigens comprise S. pneumoniae saccharide 6A.
 64. The processof any one of claims 50-63 wherein the further antigens comprise S.pneumoniae saccharide
 1. 65. The process of any one of claims 59-64wherein the further antigens comprise S. pneumoniae saccharide
 5. 66.The process of any one of claims 59-65 wherein the further antigenscomprise S. pneumoniae saccharide 7F.
 67. The process of any one ofclaims 59-66 wherein the further antigens comprise S. pneumoniaesaccharide
 3. 68. The process of any one of claims 59-67 wherein thefurther antigens comprise S. pneumoniae saccharide 23F.
 69. The processof any one of claims 59-68 wherein the further antigens comprise S.pneumoniae saccharide 6B.
 70. The process of any one of claims 59-70wherein the further antigens comprise S. pneumoniae saccharide 6B. 71.The process of any one of claims 59-70 wherein the further antigenscomprise one or more S. pneumoniae proteins selected from the groupconsisting of the Poly Histidine Triad family (PhtX), Choline BindingProtein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpXtruncate-LytX truncate chimeric proteins (or fusions), pneumolysin(Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125 and Sp133.
 72. The processof any one of claims 60-71 wherein the further antigens compriseDiphtheria Toxoid (DT), Tetanus Toxoid (TT), and either killedwhole-cell Bordetella pertussis (Pw), or two or more acellular pertussiscomponents (Pa).
 73. The process of any one of claims 60-72 wherein thefurther antigens comprise Hepatitis B surface antigen (HepB).
 74. Theprocess of any one of claims 60-73 wherein the further antigens compriseHaemophilus influenzae b (Hib) polysaccharide or oligosaccharide. 75.The process of any one of claims 60-74 wherein the further antigenscomprise one or more conjugates of a carrier protein and a capsularpolysaccharide of a bacterium selected from the group consisting of N.meningitidis type A (MenA), N. meningitidis type C (MenC), N.meningitidis type W (MenW) and N. meningitidis type Y (MenY).
 76. Theprocess of any one of claims 1-75 wherein the conjugate is mixed with anadjuvant.
 77. The process of claim 76 wherein the adjuvant is analuminium salt.
 78. The process of any one of claims 1-77 wherein theconjugate is mixed with a pharmaceutically acceptable excipient.
 79. Aconjugate obtainable by the process of any one of claims 1-78.
 80. Aconjugate obtained by the process of any one of claims 1-78.
 81. Animmunogenic composition comprising the conjugate of any one of claims79-80 and a pharmaceutically acceptable excipient.
 82. The immunogeniccomposition of claim 81 wherein the pharmaceutical excipient does notcontain a chloride salt.
 83. The immunogenic composition of any one ofclaims 81-82 wherein the pharmaceutical excipient comprises a buffer.84. The immunogenic composition of claim 83 wherein the buffer ismaleate buffer.
 85. The immunogenic composition of claim 84 whichcomprises at least one further antigen.
 86. The immunogenic compositionof claim 86 wherein the further antigens comprise S. pneumoniaesaccharides selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B,7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23Fand 33F.
 87. The immunogenic composition of any one of claims 85-86wherein the further antigens comprise S. pneumoniae saccharides 4, 9V,14, 18C, and 19F.
 88. The immunogenic composition of any one of claims85-87 wherein the further antigens comprise S. pneumoniae saccharide19A.
 89. The immunogenic composition of any one of claims 85-88 whereinthe further antigens comprise S. pneumoniae saccharide 6A.
 90. Theimmunogenic composition of any one of claims 85-89 wherein the furtherantigens comprise S. pneumoniae saccharide
 1. 91. The immunogeniccomposition of any one of claims 85-90 wherein the further antigenscomprise S. pneumoniae saccharide
 5. 92. The immunogenic composition ofany one of claims 85-91 wherein the further antigens comprise S.pneumoniae saccharide 7F.
 93. The immunogenic composition of any one ofclaims 85-92 wherein the further antigens comprise S. pneumoniaesaccharide
 3. 94. The immunogenic composition of any one of claims 85-93wherein the further antigens comprise S. pneumoniae saccharide 23F. 95.The immunogenic composition of any one of claims 85-94 wherein thefurther antigens comprise S. pneumoniae saccharide 6B.
 96. Theimmunogenic composition of any one of claims 85-95 wherein the furtherantigens comprise S. pneumoniae saccharide 6C.
 97. The immunogeniccomposition of any one of claims 85-96 wherein the further antigenscomprise one or more S. pneumoniae proteins selected from the groupconsisting of the Poly Histidine Triad family (PhtX), Choline BindingProtein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpXtruncate-LytX truncate chimeric proteins (or fusions), pneumolysin(Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125 and Sp133.
 98. Theimmunogenic composition of any one of claims 85-97 wherein the furtherantigens comprise diphtheria toxoid (DT), tetanus toxoid (TT), andeither killed whole-cell Bordetella pertussis (Pw), or two or moreacellular pertussis components (Pa).
 99. The immunogenic composition ofany one of claims 85-98 wherein the further antigens compriseHaemophilus influenzae b (Hib) polysaccharide or oligosaccharide. 100.The immunogenic composition of any one of claims 85-99 wherein thefurther antigens comprise Hepatitis B surface antigen (HepB)
 101. Theimmunogenic composition of any one of claims 85-100 wherein the furtherantigens comprise one or more conjugates of a carrier protein and acapsular polysaccharide of a bacterium selected from the group N.meningitidis type A (MenA), N. meningitidis type C (MenC), N.meningitidis type W (MenW) and N. meningitidis type Y (MenY).
 102. Theimmunogenic composition of any one of claims 85-101 further comprisingan adjuvant.
 103. The immunogenic composition of claim 102 wherein theadjuvant is an aluminium salt.
 104. A vaccine comprising the immunogeniccomposition of any one of claims 81-103.
 105. A use of the immunogeniccomposition of any one of claims 81-103 or the vaccine of claim 104 inthe prevention or treatment of bacterial disease.
 106. A use of theimmunogenic composition of any one of claims 81-103 or the vaccine ofclaim 104 in the preparation of a medicament for the prevention ortreatment of bacterial disease.
 107. A method of preventing or treatingbacterial infection comprising administering the immunogenic compositionof claims 81-103 or the vaccine of claim 104 to a patient.
 108. Anactivated bacterial saccharide, wherein the activated bacterialsaccharide comprises a repeat unit of formula (I):

wherein the activated bacterial saccharide comprises n repeat units andn is between 2 and 2400, between 500 and 2000, between 750 and 1500,between 1000 and 2000 or between 1500 and
 2300. wherein at least 0.001%but less than 50% of S1 is

and the remainder is

wherein S2 is either

and wherein S3 is either


109. The compound of claim 108 wherein less than 0.001%, 0.1%, 0.5% 1%,2%, 3%, 5%, 10%, 30% or 50% of S2 is


110. The compound of claim 108 or 109 wherein less than 0.1%, 0.5%, 1%,2%, 3%, 5%, 10%, 30% or 50% of S3 is